A novel concept for dealing with nerve agent toxicity involves taking advantage of a naturally occurring enzyme, butyrylcholinesterase (BuChE) that is able to sequester or catalyze the hydrolysis of nerve agents. This macromolecule has the potential of providing protection against a number of nerve agents with minimal side effects, since it can stoichiometrically bind and/or metabolize a nerve agent before its distribution to the site of toxic effect. The main obstacle to development of this type of therapeutic for use in humans is the need for frequent administration of a relatively short-lived product. We propose to create a novel BuChE protein that can be administered less frequently, but with greater potency, than the unmodified BuChE. We will achieve this goal using the published structural information for butylcholinesterase to rationally design polyethylene glycol (PEG)-BuChE conjugates using cysteine-reactive PEG. We will introduce a new "free" cysteine using she-directed mutagenesis in regions of BuChE that are believed to be non-essential for biological activity. The "free" cysteine residue will serve as the site for the covalent modification of the protein using a thiol-reactive PEG. This technology allows for the creation of novel, fully active PEG-Cys-BuChE analogues of defined structure and overcomes the problems of reduced bioactivity and heterogeneity when a protein is modified using a standard amine-reactive PEG. During Phase I we will identify sites in BuChE that can be modified without affecting the protein's in vitro bioactivity and further, demonstrate that PEGylation enhances BuChE's circulating half-life and efficacy in vivo. During Phase II, we develop a cost effective manufacturing process and produce sufficient quantities of the PEGylated recombinant BuChE for testing in animal toxicity models and biochemical characterization.